In Vitro and In Vivo Mutagenesis.
Directed evolution is one of the most powerful tools for engineering proteins, especially when a significant number of mutations have to be iteratively accumulated to achieve the desired phenotype (Minshull, J. & Stemmer, W. P. C. Protein evolution by molecular breeding. Curr. Opin. Chem. Biol. 3, 284-290 (1999); Petrounia, I. P. & Arnold, F. H. Designed evolution of enzymatic properties. Curr. Opin. Biotech. 11, 330 (2000)). In vitro methods for creating genetic diversity are very powerful but laborious to apply iteratively when selection has to be done on transfected cells or organisms. In vivo mutagenesis avoids repetitive transfection and re-isolation of genes but normally randomizes the entire genome wastefully rather than focusing on the gene of interest (Greener, A., Callahan, M. & Jerpseth, B. An efficient random mutagenesis technique using an E. coli mutator strain. Mol. Biotechnol. 7, 189-95 (1997)).
Polypeptide variants may provide polypeptides having improved properties as compared to the parent polypeptides. For example, a variety of Aequorea GFP-related fluorescent proteins having useful excitation and emission spectra have been engineered by modifying the amino acid sequence of a native sequence GFP from A. Victoria (see Prasher et al., Gene 111:229-233, 1992; Heim et al., Proc. Natl. Acad. Sci. USA 91:12501-12504, 1994; U.S. Pat. No. 5,625,048; International application PCT/US95/14692, now published as PCT WO96/23810, each of which is incorporated herein by reference). However, there is need for further methods for providing polypeptide variants and for improved polypeptides, including further need for fluorescent protein variants having improved properties.
Somatic Hypermutation.
B lymphocytes (B cells) can specifically mutate immunoglobulin chains through a process called somatic hypermutation (SHM). SHM uses activation-induced cytidine deaminase (AID) and error-prone DNA repair to introduce point mutations into the rearranged V regions of immunoglobulin in a rate of ˜1×10−3 mutations per base pair per generation, 106 times higher than that in the rest of the genome (Rajewsky, K., Forster, I. & Cumano, A. Evolutionary and somatic selection of the antibody repertoire in the mouse. Science 238, 1088-94 (1987)).
Our understanding of SHM has been further advanced by more recent work (see, e.g., Papavasiliou, F. N. & Schatz, D. G. Somatic hypermutation of immunoglobulin genes: merging mechanisms for genetic diversity. Cell 109 Suppl., S35-S44 (2002); Martin, A. & Scharff, M. D. AID and mismatch repair in antibody diversification. Nat. Rev. Immunol. 2, 605-14 (2002); Neuberger, M. S., Harris, R. S., Di Noia, J. & Petersen-Mahrt, S. K. Immunity through DNA deamination. Trends Biochem. Sci. 28, 305-12 (2003); Bachl, J., Carlson, C., Gray-Schopfer, V., Dessing, M. & Olsson, C. Increased transcription levels induce higher mutation rates in a hypermutating cell line. J. Immunol. 166, 5051-7 (2001); Wang, C. L., Harper, R. A. & Wabl, M. Genome-wide somatic hypermutation. Proc. Natl. Acad. Sci. U.S.A 101, 7352-7356 (2004)). Somatic hypermutation has not been used for polypeptide mutagenesis to provide polypeptide variants or improved polypeptides.